Despite significant advances in the study of oogenesis, there are major gaps in our understanding of how mammalian meiosis is regulated through protein interactions and signaling systems. Recently it has become clear that members of the 14-3-3 protein family are involved in regulating an impressive variety of cellular processes. There is evidence, mostly from studies of amphibians, that 14-3-3 proteins are central to oogenesis, particularly in oocyte maturation. Moreover, 14-3-3 proteins in somatic cells are vital regulators in a number of signal transduction pathways, cell cycle control, protein trafficking, apoptosis, and aspects of embryonic development. Yet, little is known about the functions of 14-3-3 in mammalian gametes and early embryos. The primary objectives of this project are 1) to use several techniques, including Tandem Affinity Purification, to identify the proteins that interact with 14-3-3 in oocytes, eggs and early embryos, and 2) to determine the function of 14-3-3 at these stages of development. Proteins often form assemblies and both individual and collective protein functions are dependent on these complex binding interactions. The combined techniques of affinity purification and mass spectrometry are powerful tools to purify complexes and identify interacting proteins to understand normal and abnormal cellular function. Tandem Affinity Purification (TAP) is, so far, the best protein affinity purification system because the purification is very selective and it preserves endogenous protein interactions that may be missed or obscured with other techniques. Proteins that bind to 14-3-3 will be identified in the mouse oocyte, egg and early embryo using a novel, new transgenic mouse line whose animals express a TAP-tagged 14-4-3 in these cells. Focusing on filling in some of the gaps in our understanding of mammalian oocyte maturation and early development, the role of 14-3-3 will be assessed by suppressing its ability to bind to target proteins and examining the consequences. Some of the 14-3-3 binding proteins in oocytes and eggs are known or predicted, but the TAP 14-3-3 purification will likely identify new interacting proteins. The combination of identifying interacting proteins and determining the function of such interactions will enhance our understanding of mammalian oogenesis. PUBLIC HEALTH RELEVANCE: The proposed studies are designed to uncover some of the mechanisms responsible for the normal production of reproductive cells in the ovary. These studies introduce a new method to better understand the complex interactions between proteins in oocyte, mature eggs, and early embryos. The research will provide insights into some of the processes that affect normal fertility and also infertility in mammals, including humans.